Once the genome is sequenced, the second phase of the Humane Genome Project is aimed at surveying single nucleotide polymorphisms (SNPs) that exist in different human DNAs. It is now clear that changes in sequence as small as a single base in a gene can be diagnostic for many disease states and susceptibilities. As a result, in the future the medical community will commonly make use of genetic screens of individual patients as a routine part of diagnosis. This means that the development of rapid, sensitive, and accurate methods for detecting and identifying SNPs is very important to the future of medicine.
One type of SNP that has been recognized as very important to diagnosis of cancer is the set of single-base mutations that have been associated with the development of cancer. An increasing number of point mutations in oncogenes and tumor suppressor genes have been linked to cancer, and some of these are not merely diagnostic (i.e., associated with cancer) but also causative (responsible for the cancerous phenotype). Among the most important examples already starting to be screened in medical laboratories today are point mutations in the H-ras and K-ras oncogene family as well as in the p53 tumor suppressor gene. In some kinds of cancer, such point mutations are strongly specific; for example, a single K-ras codon 12 point mutation is found in ˜90% of all pancreatic cancers.
SNPs can be detected using various DNA ligation strategies. Methods for joining strands of DNA are widely used in chemistry, molecular biology, and biomedicine. Both enzymatic and chemical methods for DNA ligation are known. Enzymatic ligation of DNA has been important for the development of DNA diagnostic methods. Moreover, current methods for enzymatic DNA ligation cannot be used for direct detection of RNAs, since these ligases require duplex DNAs as substrates. In addition, although very short probes exhibit the highest sequence specificity, ligase enzymes cannot utilize oligodeoxynucleotides shorter than about 9 nucleotides (C. Pritchard et al., Nucleic Acids Res. 25: 3403–3407 (1997)). Also, because of the sensitivity to native DNA structure, ligase-mediated approaches are unlikely to be useful with modified probes that contain nonnatural DNA structure such as PNA, phosphoramidate DNA, or 2′-O-methyl RNA. Even relatively simple modifications such as conjugation with biotin or fluorescent labels may be expected to cause difficulties near the ligation junction. Finally, ligase methods are not likely to be useful in intact cellular or tissue preparations, since it would be difficult to deliver the ligase into cells.
By comparison, nonenzymatic ligation strategies have the advantage of not requiring natural structure at the ligation site and, potentially, of proceeding in higher yields at lower cost. Some nonenzymatic ligation approaches require reducing reagents such as borohydride, oxidizing reagents such as ferricyanide, condensing reagents such as carbodiimides or cyanoimidazole, or UV irradiation to carry out the reaction. Other nonenzymatic ligations, termed autoligations or self-ligations, proceed in the absence of additional reagents. While the need for added reagents is not limiting in many situations, autoligation is simpler, and might be carried out in media where reagents are inactive or where they will affect biochemical processes.
Letsinger et al. (U.S. Pat. No. 5,476,930) have described an irreversible, nonenzymatic, covalent autoligation of adjacent, template-bound oligodeoxynucleotides wherein one oligonucleotide has a 5′ or 3′ α-haloacyl reactive group, such as a 3′-bromoacetylamino, and the second oligonucleotide has a 3′ or 5′ phosphorothioate group. The resulting linkage takes the form of a thiophosphorylacetylamino bond.
Letsinger et al. (U.S. Pat. No. 5,780,613; Herrlein et al., J. Am. Chem. Soc., 117, 10151 (1995)) have also described an approach to the templated ligation of oligodeoxynucleotides that involves reacting an oligonucleotide having a 3′-phosphorothioate group with a second oligonucleotide having as 5′ tosylate leaving group giving SN2 displacement and resulting in more natural DNA structure, having a sulfur atom replacing one of the bridging phosphodiester oxygen atoms. This method was used to ligate self-templated ends to yield dumbbell-type structures in good yields. However, due to the reactivity of the 5′-tosylate to ammonia, it was necessary to use labile protecting groups and rapid deprotection, and significant degradation was still observed for oligonucleotides carrying the reactive leaving group. Xu et al. (Tetrahedron Lett., 38, 5595–5598 (1997)) describe an improvement in this method utilizing, as the leaving group, a 5′-iodide which is stable to ammonia deprotection.
There is a clear need for simple, rapid and reliable methods for detecting SNPs, and there will be many formats in which they will be applied, such as in sequence detection in PCR-amplified DNAs, detection in DNAs or in RNAs isolated directly from clinical samples (blood, tissue, urine, etc), detection in isolated cells (such as from blood), detection in tissue cross sections (such as from biopsies), and detection in the living body. Because of the advantages of nonenzymatic ligation methods in both diagnostic and preparative nucleic acid technologies, further improvements in the speed, selectivity and specificity of nonenzymatic ligation of oligonucleotides are very important. An improved ligation chemistry (1) would require no added reagents to carry out the reaction, (2) would require no post-synthesis modification of the DNA prior to reaction, (3) could be carried out on an RNA template (unlike enzymatic ligations), and (4) would create a junction that causes little perturbation to the DNA structure.